During the interaction of CD4+ T cells with antigen-pulsed antigen presenting cells dynamic changes in the distribution of cell surface receptors and signaling intermediates occur with the T cell. The organized structures that these molecules create are referred to as "SupraMolecular Activation Cluster" (SMACs). Upon interaction with APCs not pulsed with antigen there is no formation of SMACs. The central region, the cSMAC is comprised of the TCR/CD3 complex and the signaling intermediate PKCtheta. Surrounding the cSMAC is a region referred to as the peripheral SMAC, or pSMAC, which contains LFA/1 and the cytoskeletal protein, talin. The formation of these distinct c- and p-SMACs zones suggest that the activation of T cells does not arise from the stimulation of random individual T cell receptors. This is further highlighted by the observation that the association with the cSMAC of several molecules, such as CD45 and the protein kinase p561ck, is transient. Previous studies of SMAC formation during T cells:APC interaction have focused extensively on CD4 T cells. We have obtained preliminary evidence that CD8 T cells interacting with antigen pulsed presenting cells also form cSMAC and pSMAC region. In this project we will examine, at the single cell level, the formation of the cSMAC and the pSMAC during the interaction of CD8+ cytotoxic T cells with target cells under physiological conditions. The proposed studies incorporate 4 specific aims: Aim 1: To determine whether cSMAC and pSMAC zones are established on CD8+ T cells upon interaction with targeted cells. Aim 2: To determine whether CD8, like CD4, initially clusters in cSMAC and subsequently moves to more distal regions. Aim 3: To determine whether the movement of CD8 is dependent upon it binding to MHC class I molecules on the surface of the target cell. Aim 4: To determine how supramolecular activation clusters are assembled on thymocytes undergoing selection events. These studies will provide insight into the role of the CD4 and CD8 co- receptors in the stimulation of T cells at different stages of differentiation.